The understanding of virus-host interactions, including the identity of the host factors with which viruses interact to mediate their replication and cytopathic effects, remains a challenging frontier in virology. For a number of viruses, specific host factors have been implicated in some important infection processes such as cell attachment, DNA transcription and replication, and others. However, for many important virus functions, such as genome replication by (+) strand RNA viruses, the nature of the host factors is not completely known. For most viruses, identification of relevant host factors would be greatly assisted by ready genetic dissection of the host. Particularly effective eukaryotic genetics exists for the yeast Saccharomyces cerevisiae, as illustrated by prior identification of host genes required for the maintenance of its endogenous L-A double-stranded RNA virus (Wickner, R. B., eds. Broach, J. R., Pringle, J. and Jones, E. W. (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.), pp. 263-296, 1991). S. cerevisiae has been previously shown to support the replication and assembly of other viruses endogenous to fungi (Lhoas, P. Nature-New Biology, 236:86-87, 1972; Schmitt, M. J. and Neuhausen, F., J. Virol. 68:1765-1772, 1994).
The present invention involves viruses that are non-endogenous to fungi, such as flock house virus (FHV). We show in the Examples that the yeast Saccharomyces cerevisiae can support replication of FHV resulting in the production of infectious virions.
Previously, virus-like particles (VLP) with unspecified nucleic acid content have been synthesized in yeast from the plasmid-expressed capsid proteins of other non-endogenous viruses; hepatitis B virus (Valenzuela, P., et al., Nature 298:247-350, 1982; Miyanohara, A., et al., Proc. Natl. Acad. Sci. USA 80:1-5, 1983; Kniskern, P. J., et al., Gene 46:135-141, 1986; Miyanohara, A., et al., J. Virol. 59:176-180, 1986), the potyvirus johnsongrass mosaic virus (Jagadish, M. N. et al., J. Gen. Virol. 72:1543-1550, 1991), the papillomaviruses HPV-6, HPV-16, HPV-6a, HPV-A18, and CRPV (Hofmann, K. J., et al., J. Gen. Virol. 77:465-468, 1996; and references therein), and the picornavirus polioviruis (Jore, J. P. M., et al., Yeast 10:907-922, 1994). Retrovirus human T-lymphotropic virus (HTLV-III) VLP have been produced, although VLP production was inferred from protein processing (Kramer, R. A., et al., Science 231:1580-1584, 1986). There is also a report of retrotransposon gypsy "VLP" production, although the VLPs did not resemble "normal" VLPs (Yoshioka, K., et al. FEBS Letters, 302:5-7, 1992). Spanning three of four subfamilies in the family Saccharomycetaceae, these non-endogenous VLPs have beer produced in Saccharomyces cerevisiae, Hansenula polymorpha, Yarrowia lipolytica, Schizosaccharomyces pombe, and Pichia pastoris (Shen, S.-H., et al., Gene 84:303-309, 1989; Janowicz, Z. A., et al., Yeast 7:431-443, 1991; Hamsa, P. V. and B. B. Chatto, Gene 143:165-170, 1994; Sasagawa, T., et al., Virology 206:126-135, 1995; Tschopp, J. F., et al., Biotechnology 5:479-485, 1987).
A description of increasing yields of tobacco mosaic virus (TMV) with time following incubation of S. cerevisiae spheroplasts with TMV virions (not irrefutable evidence) has been reported (Coutts, R. H. A. and Cocking, E. C., Nature 240:466-467, 1972).
Systems were recently developed that demonstrate the RNA-dependent replication, transcription and persistence in yeast of derivatives of brome mosaic virus (BMV), a plant-infecting member of the alphavirus-like superfamily (Janda, M. and Ahlquist, P., Cell 72:961-970, 1993; Quadt, R., et al., Proc. Natl. Acad. Sci. USA 92:4892-4896, 1995; for a simple review see Gallepp, G. Understanding infection, UW-Madison College of Agricultural and Life Sciences Science Report, pp. 28-29, 1996). Yeast expressing the viral components of the BMV RNA replicase support replication and gene expression by BMV RNA derivatives. Such RNAs are transmitted through mitosis, so that Ura.sup.+ strains can be obtained by transfecting Ura.sup.- yeast with a BMV RNA replicon expressing the URA3 gene. This system provides a basis for selecting mutations in host genes required for BMV RNA replication.
Thus, BMV is the first instance of genomic replication of a non-endogenous virus in S. cerevisiae. Attempts to replicate the poliovirus genome in yeast have failed (Coward, P. and Dasgupta, A., J. Virol. 66:286-295, 1992; Das, S., et al., J. Virol. 68:7200-7211, 1994).
The expression of wild-type BMV RNA3, which contains the BMV coat protein gene, has been reported in conjunction with viral replicase proteins in yeast (Quadt, R., eta. Proc. Natl. Acad. Sci. USA 92:4892-4896, 1995).
Flock house virus (FHV) belongs to the Nodaviridae: family of (+) strand RNA viruses, whose members infect invertebrates and vertebrates (for review see Miller, L. K., in Fields Virology, eds. Fields, B. N., et al. Lippincott-Raven Publishers, Philadelphia, Vol. I, pp. 549-551, 1996). Several members which infect hatchery-reared fish have recently been identified (Nishizawa, T., et al., J. Gen. Virol. 76:1563-1569, 1995).
The 4.5 kb genome of FHV is one of the smallest among animal viruses. FHV particles are infectious to insect cells, and naked FHV genomic RNA is infectious to insect, plant (Selling, B. H., et al., Proc. Natl. Acad. Sci. USA 87:434-438, 1990) and mammalian (Ball, L. A., et al., J. Virol. 66:2326-2334, 1992) cells.
An FHV plaque assay (Selling, B. H. and Rueckert, R. R., J. Virol. 51:251-253, 1984) and neutralizing antibodies (Gallagher, T. M., Ph.D. thesis (University of Wisconsin, Madison), pp. 84-111, 1987) allow sensitive detection of infectious FHV virions.
Infectious transcripts from full-length FHV cDNAs can be produced in vitro (Dasmahaptra, B., et al., Proc. Natl. Acad. Sci. USA 83:63-66, 1986) and in vivo (Ball, L. A., Proc. Natl. Acad. Sci. USA 91:12443-12447, 1994). Nodaviruses use unique regulatory cis-elements, including Nodaviral-specific RNA replication (Zhong, W., et al., Proc. Natl. Acad. Sci. USA 89:11146-11150, 1992; Ball, L. A. and Li, Y., J. Virol. 67:3544-3551, 1993; Li, Y. and Ball, L. A., J. Virol. 67:3854-3860, 1993; Ball, L. A., J. Virol. 69:720-727, 1995) and packaging signals (Zhong, W., et al., supra, 1992; Zhong, W., Ph.D. thesis (University of Wisconsin, Madison, pp. 119-138, 1993), which offer the potential for new mechanisms of RNA and protein expression regulation. FHV RNAs have been used to carry and express heterologous sequences (Zhong, supra, 1993). The ability to package these chimeric RNAs into FHV particles has been demonstrated (Zhong, supra, 1993). In addition, a replicase capable of full in vitro replication of added FHV RNA has been isolated from infected cells (Wu, S.-X. and Kaesberg, P., Virology 183:392-396, 1991; Wu, S.-X., et al., Proc. Natl. Acad. Sci. USA 89:11136-11140, 1992).
The X-ray structure of FHV (Fisher, A. J. and Johnson, J. E., Nature 361:176-179, 1993) and the related black beetle virus (Hosur, M. V., et al., Proteins: Struct. Funct. Genet. 2:167-176, 1987) have been solved at 3.0 .ANG. resolution, allowing for insertion of antigens on the particle surface.
The FHV genome is bipartite (FIG. 1A) (Scotti, P. D., et al., Arch. Virol. 75:181-190, 1983), with both RNAs packaged in the same particle (Selling, B. H. and Rueckert, R. R., supra, 1984; Gallagher, T. M., supra, 1987). RNA1 (3.1 kb) (Dasmahapatra, B., et al., J. Mol. Biol. 182:183-189, 1985) encodes all viral contributions to the FHV RNA replicase and can replicate autonomously (Gallagher, T. M., et al., J. Virol. 46:481-489, 1983). RNAL serves as mRNA for protein A (112 kDa) (Friesen, P. D. and Rueckert, R. R., J. Virol. 37:876-886, 1981), which contains the GDD amino acid sequence motif characteristic of RNA polymerases and is essential for FHV RNA replication (Ball, L. A., J. Virol. 69:720-727, 1995). An RNA1-encoded subgenomic RNA, RNA3 (0.4 kb) (Guarino, L. A., et al., Virology 139:199-203, 1984), serves as mRNA for protein B (10 kDa) (Friesen, P. D. and Rueckert, R. R., J. Virol. 42:986-995, 1982). Protein B is not required for production of (+) or (-) strand RNA1 or RNA3 (Ball, L. A., supra, 1995). RNA2 (1.4 kb) encodes the virion capsid protein precursor, .alpha. (43 kDa) (Friesen, P. D. and Rueckert, R. R., supra, 1981; Dasgupta, R. and Sgro, J.-Y., Nucl. Acids. Res. 17:7525-7526, 1989).
Before the present invention, the art of molecular biology lacked a method of producing non-endogenous particles containing nucleic acid sequences in yeast and of replicating animal virus nucleic acid sequences, most particularly Nodaviridae virus nucleic acid sequences, using non-endogenous viral nucleic acid replication proteins in yeast.